Immunochromatographic test strip and detection method using immunochromatography for detecting object in red blood cell-containing sample

ABSTRACT

A problem to be solved by the present invention is to provide an immunochromatographic test strip and a detection method using immunochromatography avoiding aggregation of colloidal gold conjugates while red blood cells in whole blood are agglutinated and then separated and removed in the case of using polybrene as a blood-agglutinating agent and the colloidal gold conjugates as a detection reagent. To solve the problem, the present inventers reviewed a past reagent configuration itself from a completely different viewpoint rather than selecting type and amount of polyanions and, as a result of extensive study on each element, the inventers surprisingly found that aggregation of colloidal gold can be suppressed by using a certain buffer solution without using neutralization by polyanions.

PRIORITY CLAIM AND CROSS-REFERENCE

This application is a Divisional of copending application Ser. No.14/389,180, filed Sep. 29, 2014, which is a U.S. national stageapplication of International Application No. PCT/JP2013/060003, filedApr. 1, 2013, which claims the benefit under 35 U.S.C. §119(a) to PatentApplication No. 2012-083165, filed in Japan on Mar. 30, 2012, thecontents of which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to an immunochromatographic test strip anda detection method using immunochromatography for detecting an object(analyte) in a red blood cell-containing sample. In particular, thepresent invention relates to a test strip treated with a certain buffersolution to avoid aggregation of colloidal gold conjugates acting as adetection reagent even when a red blood cell-agglutinating agent isused, and a detection method using the buffer solution.

BACKGROUND ART

Clinics and small hospitals recently have increasing needs for“conducting tests and knowing the result during patient consultation”and thus tests are increasingly conducted as point-of-care testing(POCT) instead of conventional outsourced examination. POCT is acollective term for tests generally conducted “near a patient” in anexamination room, a hospital ward, etc., and since a doctor can quicklyinterpret test results to take measures, POCT significantly contributesto the quality of consultation. As POCT as described above becomespopular, devices incorporating lateral flow immunochromatographic teststrips etc. are distributed as commodities in the market.

An immunochromatographic test strip enables detection of an object in asample, without the need of preparation of a reagent at the time oftesting, through only a simple operation such as directly dripping atest specimen (sample) such as blood and urine onto the test strip andis very useful for conveniently and quickly analyzing an analyte(detection object) and, therefore, a large number of the test strips areput to practical use for POCT.

The immunochromatographic test strip is generally a membrane made up ofa porous body comprising a sample-supply portion, a spreading portion,and a detecting portion and has a structure in which a labeled antibodyto an analyte is retained at a spreading start part of the spreadingportion in a dissoluble manner such that the labeled antibody can passthrough the spreading portion and reach the detecting portion aftercontact with a sample while an antibody is immobilized at a part of thespreading portion to constitute the detecting portion. When the sampleis dripped onto the sample-supply portion, if an analyte is contained inthe sample, the analyte in the sample specifically binds to the labeledantibody to form a complex and the complex spreads through the spreadingportion in the downstream direction and further binds to the immobilizedantibody. Therefore, by detecting a sandwich-type complex of the labeledantibody, the analyte, and the immobilized antibody in theantibody-immobilization part, the analyte can be qualitatively orquantitatively analyzed. An example of a label constituting a detectionreagent (conjugate) such as the labeled antibody is colloidal goldparticles and a color reaction of the colloidal gold particles enablesqualitative detection. The analyte in the sample can also quantitativelybe detected based on a degree of coloring.

If a sample is whole blood, red blood cells cannot move in a membranethat is a porous body, and will problematically clog pores of themembrane and obstruct spreading of the sample. Therefore, if whole bloodis used as a sample, red blood cells must preliminarily be separated andremoved from the whole blood and known methods include a method ofremoving red blood cells after sedimentation through centrifugationbefore measurement, and a method of filtrating and removing red bloodcells agglutinated by a red blood cell-separating agent beforemeasurement or at the start of measurement.

For example, polybrene is known as a red blood cell-separating agent(Patent Documents 1, 2, and 3).

In Patent Document 1, polybrene is described as an example of ablood-agglutinating agent made of synthetic water-soluble polymer.

In Patent Document 2, a glass-fiber blood cell-separating membrane forchromatography containing polybrene as a red blood cell-agglutinatingsubstance is described. Because passage of blood through a bloodcell-separating membrane is associated with hemolysis when polybrene issolely used, a technique is disclosed for coating a bloodcell-separating membrane with PVA for avoiding the hemolysis.

Although polybrene is generally known as a blood-agglutinating agent asdescribed above, if polybrene is used for immunochromatography employinga metal conjugate as a detection reagent, polybrene will problematicallycause not only aggregation of red blood cells in whole blood but alsoaggregation of the metal conjugate (Patent Document 3).

Patent Document 3 discloses a technique for preventing such aggregationof a metal conjugate. Briefly, disclosed is an immunochromatographyassay device that has a polycation such as polybrene as a bloodcell-separating agent bound in the upstream of chromatography carriersand a polyanion for neutralizing the polycation bound in the downstreamthereof. It is described that aggregation of a metal conjugate made ofselenium can be prevented by this technique since the electric charge ofthe polycation is neutralized by the polyanion.

CITATION LIST Patent Literature

-   Patent Document 1: JP H03-205563 A-   Patent Document 2: JP H05-099918 A-   Patent Document 3: JP 2002-509254 A

SUMMARY OF INVENTION Technical Problem

When immunochromatography was practiced using whole blood as a sampleand employing polybrene as a blood agglutinating agent and a colloidalgold conjugate as a detection reagent, the present inventers attemptedto neutralize positive electric charge of polybrene by adding polyanionsso as to prevent aggregation of the colloidal gold conjugate inaccordance with the disclosure of Patent Document 3. However, althoughthe red blood cell in the whole blood could be agglutinated and thenseparated and removed, the aggregation of colloidal gold conjugatescould not be prevented. Although the reason is uncertain, this may beattributable to the difference between selenium conjugates of PatentDocument 3 and colloidal gold conjugates, and the difference in the typeof polyanions, etc.

A problem to be solved by the present invention is to provide animmunochromatographic test strip and a detection method usingimmunochromatography avoiding aggregation of colloidal gold conjugateswhile red blood cells in whole blood are agglutinated and then separatedand removed in the case of using polybrene as a blood-agglutinatingagent and the colloidal gold conjugates as a detection reagent.

Solution to Problem

In order to investigate the method for solving said problem, the presentinventers reviewed the configuration of the existing reagent itself froma completely different viewpoint rather than the selection of type oramount of polycations that are causative agents of the problem and, as aresult of extensive study on each constituent element, the inventerssurprisingly found that there exists a buffer solution which has anability to suppress aggregation due to polybrene of colloidal goldconjugates and found that aggregation of colloidal gold conjugates canbe suppressed by using the buffer solution without relying on theneutralization of polycations by polyanions, and thereby, completing thepresent invention. In particular, the present invention has thefollowing configuration.

-   [1] An immunochromatographic test strip comprising the following    configuration,

(1) a membrane consisting of a porous body equipped with at least asample-supply portion, a spreading portion, and a detecting portion,

wherein a conjugate of an anti-analyte antibody labeled with colloidalgold is retained in a dissoluble manner in a part of the spreadingportion, and an antibody is immobilized in the detecting portion whichis a part of the spreading portion on the downstream side relative tothe conjugate-retaining part, and

(2) a buffer solution component for suppressing aggregation of theconjugate due to polybrene, wherein the component is contained at leastin a part of from the sample-supply portion to an upstream side of theconjugate-retaining part of the spreading portion so as to be able tocontact with polybrene that is a blood-agglutinating agent providedthrough the sample-supply portion, and

wherein the buffer solution component is selected from a groupconsisting of Bis-Tris, MES, and citric acid.

-   [2] The test strip of [1], wherein the sample supply portion    contains polybrene.-   [3] The test strip of [1] or [2], wherein the sample supply portion    contains the buffer solution component for suppressing aggregation    of the conjugate due to polybrene.-   [4] The test strip of any one of [1] to [3] above, wherein a    polyanion as a neutralizer neutralizing a cation of polybrene acting    as the blood-agglutinating agent is not included.-   [5] An immunochromatographic detection method, comprising the steps    of:

(A) supplying a sample to a sample-supply portion of a test stripcomprising a membrane consisting of a porous body equipped with at leasta sample-supply portion, a spreading portion, and a detecting portion,

wherein a conjugate comprising a colloidal gold sensitized with anantibody to an object is retained in a dissoluble manner in a part ofthe spreading portion, and an antibody is immobilized in the detectingportion which is a part of the spreading portion on the downstream siderelative to the conjugate-retaining part;

(B) agglutinating a blood-derived component in the sample throughcontact between polybrene and the sample in the sample-supply portion orin the upstream of the sample-supply portion;

(C) separating and removing aggregates acquired at step (B) from thesample;

(D) bringing a sample component acquired at step (C) after separationand removal of the aggregates into contact with the colloidalgold-containing conjugate, the step being performed in the presence of abuffer solution having ability to suppress aggregation of the conjugatedue to polybrene; and

(E) detecting a complex of the object in the sample component and theconjugate acquired at the step (D) in the detecting portion, and

wherein the buffer solution component is selected from a groupconsisting of Bis-Tris, MES, and citric acid.

-   [6] The detection method of [5] above, wherein the sample-supply    portion contains polybrene.-   [7] The detection method of [5] or [6] above, wherein the    sample-supply portion contains the buffer solution component for    suppressing aggregation of the conjugate due to polybrene.-   [8] The detection method of any one of [5] to [7] above, wherein a    polyanion as a neutralizer neutralizing the cation of polybrene    acting as the blood-agglutinating agent is not included.

Advantageous Effects of Invention

According to the present invention, in immunochromatography usingpolybrene as a blood-agglutinating agent and a colloidal gold conjugateas a detection reagent, red blood cells can be agglutinated and thenseparated and removed from a sample without aggregation of the colloidalgold conjugate by using a buffer solution having ability to suppressaggregation of the colloidal gold conjugate due to polybrene, by whichthe colloidal gold conjugate can play the original role of the detectionreagent and, therefore, an analyte in the sample can accurately bedetected and measured. Since addition of a polyanion as a polycationneutralizer is not an essential condition, a reagent configuration canbe made extremely simple and, therefore, the present inventionadvantageously leads to reduction of factors causing unpredictableeffect to the immune reaction and to the spread of the analyte on thestrip.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of structure of an immunochromatographic teststrip of the present invention.

DESCRIPTION OF EMBODIMENTS

(Immunochromatographic Test Strip)

An immunochromatographic test strip of the present invention is amembrane consisting of a porous body equipped with at least a“sample-supply portion”, a “spreading portion”, and a “detectingportion” and has a structure in which a labeled antibody to an analyteis retained at a spreading start part of the spreading portion in adissoluble manner such that the labeled antibody passes through thespreading portion and reaches the detecting portion after contact with asample while an antibody is immobilized at a part of the spreadingportion to constitute the detecting portion.

An example embodying these constituent elements may be a test stripincluding a sample pad playing a role of a sample-supply portion, aconjugate pad having a labeled antibody to an analyte retained in adissoluble manner and playing a role of the spreading portion, and aninsoluble membrane having an antibody immobilized at a part thereof andplaying a role of the spreading portion and the detecting portion.Therefore, a typical immunochromatographic test strip of the presentinvention has the following configuration:

(1) a sample pad to be supplied with a sample;

(2) a conjugate pad disposed in the downstream of the sample pad andretaining in a dissoluble manner a conjugate in which a first antibodyis sensitized on a colloidal gold surface; and

(3) an insoluble membrane disposed in the downstream of the conjugatepad and immobilizing a second antibody which binds to a complex of theconjugate and an analyte.

The sample pad, the conjugate pad, and the insoluble membrane mayconstitute respective different carriers, or two of the elements mayconstitute one carrier, and may take any form as long as the sample pad,the conjugate pad, and the insoluble membrane are arranged in this orderfrom the upstream toward the downstream.

The immunochromatographic test strip may be a strip further having anyone or more of an absorption pad and a 3rd pad disposed and mountedalong with the above constituents. The test strip is usually disposed ona solid phase support such as a plastic adhesive sheet. It is obviousthat the solid phase support is made of material not hindering thecapillary flow of the sample and that an adhesive component is made ofmaterial not hindering the capillary flow of the sample. The test stripcan be laminated with a polyester film etc., for the purpose ofincreasing the mechanical strength of the antibody-immobilized membraneand preventing evaporation (drying) of water during an assay.

(Blood-Agglutinating Agent)

A blood-agglutinating agent used in the present invention is preferablypolybrene. Polybrene has a chemical name of hexadimethrine bromide andis one of cationic polymers.

In the present invention, polybrene is used for agglutinating red bloodcells in whole blood used as a sample. With regard to the manner ofusing polybrene, polybrene may be added to a diluent which dilutes thesample or may directly be added to the sample and, in other cases,polybrene may be contained in the sample-supply portion (sample pad) ofthe immunochromatographic strip. In such usage forms, polybrene comesinto contact with whole blood and the red blood cells in the whole bloodare agglutinated. The aggregates of red blood cells are removed throughsome kind of filtration, and are removed in the immunochromatographicstrip by the sample-supply portion on which a large portion of theaggregates remains when the sample passes through a filter constitutingthe sample-supply portion. In the present invention, a 3rd pad (bloodcell-separating membrane) described later is desirably used together soas to remove the aggregates in the sample-supply portion and morecertainly reduce the aggregates supplied to the spreading portion.

The addition amount of polybrene may be any amount capable ofagglutinating, separating, and removing red blood cells of the wholeblood so that an analyte in the whole blood sample can spread in adesirable manner and, for example, if polybrene is contained in thesample pad, the concentration is preferably equal to or greater than0.25% and more preferably 0.25 to 2% relative to the fluid volume of thesample to be dropped. In terms of manufacturing of the sample pad, theconcentration in the solution impregnated into the pad is preferablyequal to or greater than 0.5% and more preferably 0.5 to 4%.

Although polyanions are not included in the present invention unlikePatent Document 3 as a neutralizer for neutralizing the cation ofpolybrene acting as the blood-agglutinating agent, the presence ofpolyanions is not excluded as long as the polyanions are normally usedas needed within a range not affecting a reaction system withoutdeparting from the object of the present invention.

(Certain Buffer Solution)

Although a blood-agglutinating agent (polybrene) is used for the purposeof agglutinating red blood cells in a whole blood sample in the presentinvention, polybrene has a problem of causing aggregation of not onlythe red blood cells but also colloidal gold conjugates. In the presentinvention, a buffer solution must be used so as to avoid suchaggregation of the colloidal gold conjugates.

It is essential to use one or more buffer selected from a groupconsisting of Bis-Tris, MES, and citric acid as the buffer solution usedin the present invention. Bis-Tris or MES has an ability to suppress thecolloidal gold-conjugate aggregation due to polybrene at pH lower thanpKa and citric acid has an ability to suppress the colloidalgold-conjugate aggregation due to polybrene in a wide pH range of pH 4to 6.5. This is preferable not only in terms of suppression ofaggregation due to polybrene but also in terms of buffer solution inimmunoassay. Although not bound to any particular theory, all the buffersolution components are effective at pH equal to or lower than pKa and,therefore, the effect is considered as being derived from other than thebuffering capacity. It is surprising that a widely used buffer solutioncomponent may selectively agglutinate only one of the two kinds ofpolyanions, i.e., the colloidal gold conjugates and the red blood cells,and may suppress the aggregation of the other in the coexistence state.

The addition amount of these buffer solution components in the buffersolution may be any amount capable of suppressing the aggregation of thecolloidal gold conjugates due to polybrene and can experimentally beset. For example, in both a sample pad and a conjugate pad, the additionamount is equal to or greater than 20 mmol/L and preferably 20 to 100mmol/L in solution impregnated in the pads.

If the concentration of the buffer solution component is unnecessarilyhigh, hemolysis (destruction of red blood cells) may occur, which is notonly inconvenient for immunochromatography but also unsuitable for theobject of the present invention. Also from this viewpoint, the additionamount is preferably experimentally set.

A usage form of the certain buffer solution of the present invention maybe any form capable of suppressing the aggregation of colloidal goldconjugates due to polybrene used as a blood-agglutinating agent, and thecertain buffer solution may be impregnated in at least a portion of theimmunochromatographic test strip from the sample-supply portion to theconjugate-retaining part of the spreading portion to allow contact withthe polybrene component. Therefore, a certain buffer solution may beimpregnated in only the sample-supply portion or in the entire part offrom the sample-supply portion to the spreading portion. The form of thesample supply portion and/or the conjugate pad impregnated with a buffersolution includes a form in which pads are impregnated with liquidbuffer solution as well as a form in which pads are dried after the padsare impregnated with the buffer solution such that the buffer solutioncomponent is attached to the pads in a dry state.

The certain buffer solution of the present invention is usable as asample diluting solution.

Although the certain buffer solution must be used in a certain usageform in the present invention as described above, this does not preventuse of another buffer solution other than those described above foranother purpose, or use of the certain buffer solution in another usageform other than those described above. For example, when from thesample-supply portion to a conjugate immobilization part of thespreading portion are impregnated with a certain buffer solution whilean antibody-immobilization part of the spreading portion is impregnatedwith another buffer solution other than those described above, this formis obviously included within the scope of the present invention.

(Colloidal Gold)

Colloidal gold used as a label in the present invention may be anycolloidal gold capable of forming a conjugate through sensitization with(immobilization of) an antibody and capable of playing a role of a labelin a method of detecting an object (antigen) in a sample through contactwith the sample.

With regard to colloidal gold, it is considered that the colloidal goldof the present invention includes colloidal platinum as well ascolloidal gold.

The particle diameter of colloidal gold particles is known tosignificantly affect the measurement sensitivity and, for example, whenthe colloidal gold particles are retained and used in animmunochromatographic test strip, the particle diameter of colloidalgold particles is preferably 20 to 60 nm, more preferably 30 to 50 nm,and particularly preferably 40 nm. The colloidal gold can bemanufactured with a generally known method, for example, by dripping andstirring a trisodium citrate aqueous solution or a triammonium citrateaqueous solution in a heated tetrachloroauric(III) acid aqueoussolution. In this description, colloidal gold is also referred to as acolloidal gold particle(s), which has the same meaning.

(Sensitization of Colloidal Gold with Antibody)

The sensitization of colloidal gold with an antibody to an analyte isgenerally achieved by immobilization such as physical adsorption. Inthis case, the concentration of antibody is preferably prepared at aconcentration of 1 to 5 μg/mL buffer solution. The type and pH of thebuffer solution are preferably 2 mmol/L phosphate buffer solution (pH6.5 to 8) or 2 to 10 mmol/L Tris buffer solution (pH 7 to 9) and morepreferably 2 to 10 mmol/L Tris buffer solution (pH 7 to 7.5); however,another buffer solution may be used without any limitation to thoselisted above. In this description, a “conjugate” refers to colloidalgold as described above sensitized with an antibody to an analyte orwith a control antibody (or antigen).

(Blocking)

The conjugate of the present invention can be blocked by a blockingagent in a region of a colloidal gold surface not bound to an antibody.

The blocking agent of the colloidal gold conjugate is generally acomponent derived from an organism and the component derived from anorganism may be any component derived from an organism and having ablocking effect and may be an animal protein or a peptide derived froman animal protein. For example, the component derived from an organismmay be bovine serum albumin or BSA, Blocking Peptide Fragment(manufactured by TOYOBO) derived from microorganisms, NEO PROTEIN SAVER(manufactured by TOYOBO) derived from silk protein (hydrolysate ofsericin), Starting Block™ (PBS) Blocking Buffer (manufactured byPIERCE), StabilCoat™ (manufactured by SurModics), and casein.

The concentration of a component derived from an organism mayappropriately be determined depending on the component to be used. Forexample, after antibody solution is added to and mixed with colloidalgold solution adjusted to 1 OD/mL, the component derived from anorganism is added to the mixture solution to a final concentrationwithin a range of 0.1 to 10% for blocking and is more preferably usedwithin a range of 0.2 to 5%.

Alternatively, both a component not derived from an organism and acomponent derived from an organism can be mixed and used as a blockingagent of colloidal gold.

(Detection Reagent)

In the present invention, specifically, a “detection reagent” is asolution containing at least a conjugate.

The detection reagent may contain, for example, one or more stabilizers,solubilizers, etc., for the purpose of maintaining the conjugate in astable state so as to facilitate the specific reaction between theantibody immobilized to the conjugate and the analyte or to make theconjugate dissolved and fluidized promptly and effectively when mixedwith the sample. The stabilizers, solubilizers, etc. can include bovineserum albumin (BSA), sucrose, casein, and amino acids, for example.

The detection reagent may contain a known sensitizer such as2-methacryloyloxyethyl phospholylcholine for the purpose of improvementin detection sensitivity as needed.

The detection reagent may also contain EDTA or EGTA that is a chelatingagent of Ca²⁺ ions as needed.

The term “detection” or “measurement” as used herein must be construedin the broadest sense including verification and/or quantification ofthe presence of the analyte and must not be construed in a limitedmanner in any sense.

(Diluting Solution)

A diluting solution can be used in the present invention if dilution ofa sample is required depending on the concentration of an analyte in thesample. The diluting solution may be a diluting solution of anycomposition as long as the diluting solution does not significantlyinhibit the antigen-antibody reaction or, conversely, does notsignificantly facilitate the reaction resulting in excessiveagglutination causing a defect of spread by capillarity, and does notmake the antigen concentration-dependent signal detection of theantigen-antibody reaction impossible.

The diluting solution having such an effect may be, for example,purified water, saline, and a low-concentration buffer solution at pH6.0 to 10.0, for example, 10 to 20 mmol/L phosphate buffer solution, 10to 20 mmol/L Tris-HCl buffer solution, and 10 to 20 mmol/L Bis-Trisbuffer solution. A surfactant can be added to these diluting solutionsso as to control the spread rate of the sample in the strip.

The diluting solution of the present invention can contain polybrene asa blood-agglutinating agent as described above.

The diluting solution of the present invention can also contain a buffersolution component having an ability to suppress the aggregation ofcolloidal gold conjugates due to polybrene as described above. In thiscase, the buffer solution of the present invention is used as a dilutingsolution in place of the buffer solutions described above.

(Sample Pad)

In the present invention, a “sample pad” is a part playing the role of asample-supply portion receiving a sample and is shaped into a pad toabsorb a liquid sample, including any material and form allowing thepassage of liquid and the component of the analyte.

The sample pad of the present invention can contain theblood-agglutinating agent as described above. In this case, theblood-agglutinating agent can be contained at least in a part orentirety of the sample pad.

The sample pad of the present invention can contain the certain buffersolution component as described above. In this case, the certain buffersolution component can be contained at least in a part of or entirety ofthe sample pad. If the sample pad of the present invention contains boththe blood-agglutinating agent and the certain buffer solution, thesample pad may contain both at the same part or may contain atrespective different parts to avoid coexistence. The sample pad mayentirely contain both.

Specific examples of materials suitable for the sample pad include, butnot limited to, glass fiber, acrylic fiber, hydrophilic polyethylenematerial, dry paper, paper pulp, fabric, etc. A glass fiber pad ispreferably used. The sample pad may additionally be given a function ofa conjugate pad described later. The sample pad may contain a commonlyused blocking reagent as needed within a range not affecting thereaction system and not departing from the object of the presentinvention.

(Conjugate Pad)

In the present invention, a “conjugate pad” refers to a pad acquired byimpregnating a material suitable for the conjugate pad described laterwith a detection reagent specifically reactive with the analyte followedby drying. The conjugate pad has a function of allowing the detectionreagent and the analyte to form a complex when the sample passes throughthe conjugate pad. The conjugate pad may by itself be disposed incontact with an antibody-immobilized membrane. Alternatively, theconjugate pad may be disposed in contact with a sample pad so as toreceive the sample which has passed through the sample pad by acapillary flow and then transfer the sample by a capillary flow toanother pad (hereinafter also referred to as a “3rd pad”) in contactwith the surface different from the contact surface for the sample pad.The selection of one or more parts of the sample pad and the conjugatepad and how the selected parts are disposed on the antibody-immobilizedmembrane may be changed as needed.

The conjugate pad of the present invention can contain the certainbuffer solution component as described above. In this case, the certainbuffer solution component is contained at least in a part of theupstream side from a part to which the conjugate is immobilized and maybe contained in entirety of the conjugated pad.

Materials suitable for the conjugate pad include, but not limited to,paper, a cellulose mixture, nitrocellulose, polyester, acrylonitrilecopolymer, glass fiber, and nonwoven fiber such as rayon. A glass fiberpad is preferably used.

The conjugate pad may contain, as needed, a “control reagent” forsecuring reliability of immunochromatography, for example, an antibodylabeled with a label and not reactive with the sample component, or ahighly antigenic protein such as KLH (keyhole limpet hemocyanin) labeledwith a label. These control reagents are components (substances) havingno possibility of being present in the sample and can appropriately beselected.

(3rd Pad)

In the present invention, a 3rd pad can be disposed for the purpose ofremoving components unnecessary for detection of the analyte out of thecomponents in the sample reacted with the detection reagent so thatcomponents necessary for reaction can smoothly spread in the insolublemembrane to which an antibody is immobilized.

For example, blood cells and insoluble blood cell fractures aredesirably removed as the components unnecessary for detection. The 3rdpad may also be given an additional effect of preliminarily removingaggregates grown to such a size as to prevent the movement to and thesmooth spread in the antibody-immobilized membrane out of aggregatesgenerated by antigen-antibody reactions. The 3rd pad may be made of anymaterial and in any form allowing the passage of liquid and thecomponent to be detected. Specific examples are, but not limited to,glass fiber, acrylic fiber, hydrophilic polyethylene material, drypaper, paper pulp, fabric, etc.

In the present invention, a blood cell-separation membrane is desirablyused for certainly separating and removing blood cells not completelyremoved only by the blood-agglutinating agent and the sample paddescribed above.

(Immobilization of Antibody to Insoluble Membrane)

In the immunochromatographic reagent of the present invention, anantibody to the analyte can be immobilized to an insoluble membrane witha generally well-known method. For example, in the case of theflow-through format, the antibody is prepared at a predeterminedconcentration and a certain amount of the solution thereof is applied tothe insoluble membrane in a shape of a certain symbol such as dot or“+”. In this case, to secure reliability of immunochromatography, a“control line” is generally formed by immobilizing a protein or acompound capable of binding to the conjugate to a position differentfrom the antibody to the analyte. The “control line” may be formed byimmobilizing the antibody to the control reagent to a position differentfrom the antibody to the analyte.

In the case of a lateral-flow format, an antibody is prepared at apredetermined concentration and the solution thereof is applied to aninsoluble membrane in a line shape by using a device having a mechanismcapable of horizontally moving while discharging the solution from anozzle at a constant rate. In this case, the concentration of theantibody is preferably 0.1 to 5 mg/mL and more preferably 0.5 to 3mg/mL. An immobilized amount of the antibody on the insoluble membranecan be optimized by adjusting the application amount dripped onto theinsoluble membrane in the case of the flow-through format, and can beoptimized by adjusting the discharge rate from the nozzle of the devicein the case of the lateral-flow format. Particularly in the case of thelateral-flow format, 0.5 to 2 μL/cm is preferable. In the presentinvention, a “flow-through membrane assay” refers to a format in whichthe sample liquid etc. spread to perpendicularly pass through theinsoluble membrane and a “lateral-flow membrane assay” refers to aformat in which the sample liquid etc. spread to move in parallel withthe insoluble membrane.

In the present invention, the position of application to the insolublemembrane of an antibody to an analyte may be placed such that thedetection reagent spreads from the conjugate pad by capillarity andsequentially passes through the lines to which the respective antibodiesare applied in the case of the lateral-flow format. Preferably, the lineformed by applying an antibody to an analyte is located upstream whilethe line formed by applying a control antibody is located downstreamthereof. In this case, a sufficient distance is desirably placed betweenthe respective lines such that signals of labels can be detected. In thecase of the flow-through format, the position of application of anantibody to an object shall be placed such that signals of labels can bedetected.

An antibody solution applied to the insoluble membrane can normally beprepared by using a predetermined buffer solution. The types of thebuffer solution may include commonly used buffer solutions such asphosphate buffer solution, Tris buffer solution, and Good's buffersolution. The buffer solution preferably has pH in a range of 6.0 to 9.5and may appropriately be set depending on the property of the antibodyto be used. For example, a buffer solution of pH 7.2 is usable for ananti-H-FABP monoclonal antibody described later. The buffer solution maycontain salts such as NaCl, stabilizer and preservative such as sucrose,and antiseptic such as ProClin. The salts include those contained foradjusting ionic strength, such as NaCl, as well as those added at thestep of adjusting pH of the buffer solution, such as sodium hydroxide.

After an antibody is immobilized to an insoluble membrane, a blockingcan be performed by using a commonly used blocking agent in a solutionor in a vapor state to coat a portion other than theantibody-immobilized parts.

In this description, an insoluble membrane having an antibodyimmobilized as described above is also referred to as an“antibody-immobilized membrane”.

(Insoluble Membrane)

In the present invention, an insoluble membrane (hereinafter also simplyreferred to as a membrane) may be of any material. For example, thematerials include, but not limited to, polyethylene, polyethyleneterephthalate, nylons, glass, polysaccharide such as cellulose andcellulose derivatives, or ceramics. Specific examples includeglass-fiber filter-papers and cellulose filter-papers available fromMerck, Toyo Roshi, and Whatman. The pore diameter, structure, etc. of aninsoluble membrane can appropriately be selected to control the speed offlow of the immune complex of a colloidal gold-labeled antibody and anobject through the membrane. The amount of a labeled antibody binding toan antibody immobilized to the membrane can be adjusted by controllingthe speed of flow through the membrane, and therefore, the pore diameterand the structure of the membrane are desirably optimized inconsideration of combinations with the other constituent materials ofthe immunochromatographic test strip of the present invention.

(Absorption Pad)

In the present invention, an absorption pad refers to a liquid-absorbingpart absorbing the sample which has moved on and passed through theinsoluble membrane to control the spread of the sample. The absorptionpad may be disposed at the most downstream portion of the stripconfiguration in the lateral-flow format, and may be disposed on, forexample, the lower portion of the antibody-immobilized membrane in theflow-through format. For example, the absorption pad may be made offilter paper; however, this is not a limitation. Preferably, 740-E ofWhatman etc. is used.

(Detection Device)

The immunochromatographic test strip of the present invention can beinstalled and used in a container (housing) which is appropriate withrespect to the size of the strip, the method and position of addition ofthe sample, the immobilization position of antibody onantibody-immobilized membrane, the signal detection method, etc., andsuch an installed state is referred to as a “device”.

(Others)

In this description, an “insoluble membrane” is also referred to as a“solid phase” and, allowing, or a state of allowing, the insolublemembrane to physically or chemically support antigens or antibodies maybe expressed as “immobilization”, “immobilized”, “solid-phased”,“sensitize/sensitization”, or “adsorb/adsorption”.

(Sample)

In the detection method of the present invention, a “sample” containingan analyte is liquid containing a particulate component having amultiplicity of negative charges on the surface and biological samplescan include liquid containing red blood cells and particularly includewhole blood, red blood cells separated by centrifugation, etc.

(Analyte)

The analyte of the present invention is a substance present in abiological sample such as blood (whole blood), red blood cells, serum,plasma, urine, saliva, or sputum and is exemplarily illustrated as, forexample, inflammation related markers such as CRP (C-reactive protein),IgA, IgG, and IgM, coagulation/fibrinolysis markers such as fibrindegradation products (e.g., D-dimer), soluble fibrin, TAT(thrombin-antithrombin complex), and PIC (plasmin-plasmin inhibitorcomplex), circulation related markers such as oxidized LDL, BNP (brainnatriuretic peptide), and H-FABP (heart-type fatty acid-bindingprotein), metabolism related markers such as adiponectin, tumor markerssuch as CEA (carcinoembryonic antigen), AFP (α-fetoprotein), CA19-9,CA125, PSA (prostate-specific antigen), infection related markers suchas HBV (hepatitis B virus), HCV (hepatitis C virus), Chlamydiatrachomatis, and gonococcus, allergen-specific IgE (immunoglobulin E),hormones, and drugs. Among these, preferable objects are D-dimer, CRP,BNP, H-FABP, etc., for which whole blood is in high demand as a sample.

(Antibody Used in the Present Invention)

The antibody to the analyte used in the present invention is not limitedin any way by the producing method as long as the antibody isspecifically reactive to the analyte, and may be a polyclonal antibodyor a monoclonal antibody. A hybridoma producing the antibody cangenerally be prepared by cell fusion between spleen cells of an animalimmunized by using the analyte as an immunogen and myeloma cells fromthe same species in accordance with the method of Kohler and Milstein(see Nature, Vol. 256, p. 495, 1975).

When monoclonal antibodies are antibodies used in a measurement methodof detecting the analyte through formation of so-called sandwich, therelationship between a label-immobilized antibody (first antibody) andan insoluble membrane-immobilized antibody (second antibody) is suchthat the epitope of the second antibody is different from the firstantibody if the epitope of the first antibody is monovalent, and theepitope of the second antibody may be the same as or different from thefirst antibody if the epitope of the first antibody is multivalent.

In Example 1 described later, an anti-H-FABP monoclonal antibody wasused. Although a method of preparing the anti-H-FABP monoclonal antibodyused in the present invention is as described in the next section, thisis not a limitation of the present invention and a commerciallyavailable H-FABP monoclonal antibody may also be used. Examples of thecommercially available H-FABP monoclonal antibodies include clones #5B5,#10E1, etc. of HyTest and clones #M79188, #M79189, etc. of Fitzgerald(It is noted that monoclonal antibodies may be denoted by clone names ofhybridomas producing the respective antibodies for convenience. The sameapplies hereinafter).

(Example of Preparation of Anti-H-FABP Monoclonal Antibody)

(1) Preparation of Hybridoma

Human purified H-FABP (manufactured by HyTest) dissolved in PBS was usedas an immunogen. This immunogen was mixed and emulsified with completeFreund's adjuvant (manufactured by Wako Pure Chemical Industries) at aratio of one to one in fluid volume to prepare emulsion having theH-FABP concentration of 0.5 mg/mL, and 100 μL of the emulsion wassubcutaneously administered to a six-week-old female BALB/C mouse.Subsequently, 100 μL of emulsion having the H-FABP concentration of 0.2mg/mL was additionally administered three times in two and a halfmonths, and 100 μL of 0.2 mg/mL human purified H-FABP dissolved in PBSwas subcutaneously administered after 10 days from the third additionaladministration. The spleen, the groin lymph nodes, and the iliac lymphnodes were excised three days later, and the acquired spleen and lymphnode cells were mixed with myeloma cells SP2/O—Ag14 at a ratio of six toone for cell fusion in the presence of 50% polyethylene glycol 1540(manufactured by Wako Pure Chemical Industries). The fused cells weresuspended in HAT medium at 2.5×10⁶/mL as spleen cells and dispensed by0.2 mL to 96-well culture plate (manufactured by CORNING). The cellswere cultured in a 5% CO₂ incubator at 37 degrees C. and, after aboutone week, strains which produce antibodies reactive to H-FABP wereselected by using the ELISA method over the culture supernatant of wellswith growing hybridomas. Specifically, first, IgG in each of the culturesupernatants was solid-phased via goat anti-mouse IgG (Fc) antibodies(manufactured by JACKSON) on a microplate (manufactured by NUNC) andthen reacted with H-FABP. Subsequently, reaction was performed withbiotin-labeled anti-H-FABP rabbit polyclonal antibodies (manufactured byProteintech Group) and also with peroxidase-labeled streptavidin(manufactured by PIERCE). A peroxidase substrate solution containingortho-phenylenediamine (manufactured by Tokyo Chemical Industry) wasadded for coloring and, after adding 1.5 N sulfuric acid to stop thecoloring, measurement was performed by a microplate reader (Abs. 492 nm)to select hybridomas exhibiting high reactivity to H-FABP. Selectedhybridomas were cloned with a limiting-dilution method to establish tentypes of anti-H-FABP monoclonal antibody-producing hybridomas.

(2) Preparation of Monoclonal Antibody

The hybridomas acquired in (1) were intraperitoneally administered in anamount of 0.2×10⁵ cells to a 12-week-old female BALB/C mousepreliminarily intraperitoneally injected with 0.5 mL of pristane twoweeks before. The ascites was collected after about 14 days, andsupernatant was acquired by centrifugation. The supernatant was mixedwith the same amount of adsorption buffer solution (3 mol/L NaCl-1.5mol/L Glycine-NaOH, pH 8.5) and then filtrated. After the filtrate waspassed through a protein A column (manufactured by GE Healthcare)equilibrated with the adsorption buffer solution to adsorb antibodies inthe filtrate with the column, the antibodies were eluted with 0.1 mol/Lcitrate buffer solution (pH 3.0) and the anti-H-FABP monoclonalantibodies (Clone #87203 and Clone #87212) were purified.

(Measurement)

A method of quantifying signals derived from colloidal gold may beperformed in accordance with a known method and absorbance or reflectedlight intensity may be measured. Alternatively, the changes inabsorbance or reflected light intensity can be extrapolated to thecalibration curve of samples with known concentrations to measure theconcentration of the object.

(Detection Method Using Immunochromatography)

The detection method using immunochromatography of the present inventionis a method comprising at least the following steps of (A) to (E) and istypically a detection method using the immunochromatographic test stripdescribed above:

(A) a step of supplying a sample to a sample-supply portion of a teststrip comprising a membrane consisting of a porous body equipped with atleast a sample-supply portion, a spreading portion, and a detectingportion,

wherein a colloidal-gold-labeled antibody to an analyte (conjugate) isretained in a dissoluble manner in a part of the spreading portion, andan antibody is immobilized in the detecting portion which is a part ofthe spreading portion on the downstream side relative to theconjugate-retaining part;

(B) a step of agglutinating a blood-derived component in the samplethrough contact between polybrene and the sample in the sample-supplyportion or in the upstream of the sample-supply portion;

(C) a step of separating and removing aggregates acquired at step (B)from the sample;

(D) a step of bringing a sample component acquired at step (C) afterseparation and removal of the aggregates into contact with a colloidalgold-containing conjugate, the step being performed in the presence of abuffer solution having ability to suppress aggregation of the conjugatedue to polybrene; and

(E) a step of detecting a complex of the object in the sample componentand the conjugate acquired at the step (D) in the detecting portion.

EXAMPLES

The present invention will specifically be described by examples;however, the scope of the present invention is not limited by theexamples.

Example 1 Measurement Using Buffer Solution of the Present Invention (1)

1. Production of Immunochromatographic Device of the Present Invention

1) Preparation of Colloidal Gold-Labeled Anti-H-FABP Monoclonal Antibody(Anti-H-FABP Antibody Conjugate)

(i) Preparation of Colloidal Gold Solution

To 500 mL of purified water heated to 73 degrees C., 1 mL of a 5% (w/v)triammonium citrate aqueous solution was added and then stirred andmixed. Subsequently, 1 mL of a 5% (w/v) tetrachloroauric(IIl) aqueoussolution was added and reacted for 10 minutes with stirring and thereaction solution was then boiled. Subsequently, the reaction solutionwas cooled in ice water and a solution of colloidal gold with an averageparticle diameter of 40 nm was prepared. This solution of colloidal goldhaving an average particle diameter of 40 nm was adjusted to 1 OD/mLwith a 20 mmol/L Tris buffer solution (pH 7.0).

(ii) Preparation of Anti-H-FABP Antibody Conjugate

To 20 mL of the 1 OD/mL colloidal gold solution (pH 7.0), 1 mL of theanti-H-FABP monoclonal antibody (Clone #87212) diluted to 46.2 μL/mL bya 2 mmol/L Tris buffer solution (pH 7.0) was added and stirred at roomtemperature for 10 minutes. To the mixture solution of the colloidalgold and the antibody, 1 mL of a 2 mmol/L Tris buffer solution (pH 7.0)containing a 0.2% (w/v) blocking agent (NEO PROTEIN SAVER: TOYOBOBiochemical, No. NPS-301) was added and stirred at room temperature for5 minutes. The mixture solution was then centrifuged at 10 degrees C. at11900×g for 45 minutes. After supernatant was removed, 1 mL of aconjugate-diluting solution (Conjugate Dilution Buffer: SCRIPPS, No.B0221) was added to the acquired sediment to suspend the conjugate toacquire the anti-H-FABP antibody conjugate.

(iii) Preparation of Colloidal Gold-Labeled KLH (KLH Conjugate) forControl Line

To 20 mL of the 1 OD/mL colloidal gold solution (pH 6.1), 1 mL of KLH(manufactured by Sigma) diluted to 620 μg/mL by a 2 mmol/L phosphatebuffer solution (pH 6.1) was added and stirred at room temperature for10 minutes. To the mixture solution of the colloidal gold and the KLH, 1ml of a 10% bovine serum albumin (BSA) aqueous solution was added andstirred at room temperature for 5 minutes. The mixture solution was thencentrifuged at 10 degrees C. at 11900×g for 45 minutes. Aftersupernatant was removed, 1 mL of the conjugate-diluting solution wasadded to the acquired sediment to suspend the conjugate to acquire theKLH conjugate.

2) Production of Conjugate Pad

The anti-H-FABP antibody conjugate and the KLH conjugate prepared in 1),respectively, were mixed to produce conjugate solutions of 16 OD/mL and4.5 OD/mL with purified water or the following buffer solutions each ofthe water and buffer solutions containing 2.5% NEO PROTEIN SAVER and2.4% lactose, and then, a glass fiber pad having a certain volume (PallCorporation, No. 8964) was impregnated with 1.2 volumes of the conjugatesolutions relative to the volume of the pad. The pad was heated anddried at 70 degrees C. for 45 minutes in a dry oven to acquire aconjugate pad. If an additive such as a sensitizer is added as needed, anecessary amount may be added to the conjugate solutions beforeperforming the same operation.

3) Production of Anti-H-FABP Monoclonal Antibody-Immobilized Membrane

The anti-H-FABP monoclonal antibody (Clone #87203) was diluted with a 10mmol/L phosphate buffer solution (pH 7.2) containing 2.5% sucrose andprepared at 3 mg/mL, and a rabbit anti-KLH polyclonal antibody(manufactured by Bethyl) was diluted with a 10 mmol/L phosphate buffersolution (pH 7.2) containing 2.5% sucrose and prepared to 0.5 mg/mL forthe purpose of coloring of the control line. The anti-H-FABP monoclonalantibody was applied onto a nitrocellulose membrane (Merck, HF180, 260mm×25 mm) at a position inside one end of a short side and the anti-KLHpolyclonal antibody was applied at an interval of about 5 mm from theposition above by using an immunochromatography dispenser “XYZ3050” (B10DOT) set to 1 μL/cm in a line shape. The membrane was dried at 70degrees C. for 45 minutes in a dry oven to acquire anantibody-immobilized membrane.

4) Production of Sample Pad

A glass fiber pad (Lydall) cut to a necessary size as needed wasimpregnated with the same buffer solution (or purified water) as for theconjugate pad but containing 1% polybrene at 1.15 volumes relative tothe volume of the pad and was dried at 70 degrees C. for 45 minutes in adry oven, and the pad was used as a sample pad.

5) Production of Immunochromatographic Test Strip

To a plastic adhesive sheet (a), the antibody-immobilized membrane (b)was affixed such that antibody-application sites were arranged in theorder of the anti-H-FABP antibody (c) on the upstream side of spread andthen the anti-KLH antibody (d), and a blood cell-separation film (3rdpad) (e) was further mounted. The conjugate pad (f) produced in 2) wasthen disposed and mounted and the sample pad (g) produced in 4) wasdisposed and mounted to overlap the conjugate pad while the absorptionpad (h) was disposed and mounted on the end of the other side. Theimmunochromatographic test strip was produced by cutting into astructure having the constituting elements overlapped with each other asdescribed above. The test strip was installed in a dedicated plastichousing (having a sample-addition window and a detection window notdepicted in FIG. 1) at the time of an assay to implement a form of animmunochromatographic test device. FIG. 1 is a schematic of thestructure of the immunochromatographic test strip.

2. Test Results

Study on Type of Buffer Solution Applied to Sample Pad

Each immunochromatographic test strip using a sample pad and a conjugatepad impregnated with each of the nine buffer solutions or purified watermentioned above was used for measuring the level of aggregation of theconjugate after contacting with polybrene and completing the spread overa predetermined time. A plasma sample collected and centrifuged in anEDTA-2Na blood-collecting tube (Terumo Corporation) was used as a sampleinstead of whole blood so as to more clearly reveal the colloidal goldconjugate-aggregation effect of polybrene, and the H-FABP antigen(HyTest) was added. To the sample pad at the window of the test device,120 μL of the sample was added, and an overlapping portion between theblood cell-separation film (e) and the antibody-immobilized membrane (b)of the test device was uncovered 10 minutes later to measure thereflected light intensity by using the immunochromatography readerICA-1000 (Hamamatsu Photonics). The measured reflected light intensity

was indicated as aggregation sensitivity with “−” indicative ofsensitivity equal to or less than 5 mAbs., “±” indicative of sensitivityexceeding 5 mAbs. and equal to or less than 10 mAbs., and “+” indicativeof sensitivity exceeding 10 mAbs. The aggregation of colloidal goldparticles cannot be confirmed visually at 10 mAbs. or less.

The results are described in Table 1 along with results of Example 2.

Example 2 Measurement Using Buffer Solution of the Present Invention (2)

A test was conducted for the buffer solution component of Example 1 byusing materials different from Example 1.

1. Production of Immunochromatographic Device of the Present Invention(2)

1) Preparation of Colloidal Gold-Labeled Anti-D-Dimer MonoclonalAntibody (Anti-D-Dimer Antibody Conjugate)

(i) Preparation of Colloidal Gold Solution (2)

To 500 mL of purified water heated to 65 degrees C., 0.7 mL of anaqueous solution acquired by dissolving triammonium citrate andtrisodium citrate dehydrate both at a ratio of 7% (w/v) was added andthen stirred and mixed. Subsequently, 1 mL of a 7% (w/v)tetrachloroauric(III) aqueous solution was added and reacted for 10minutes with stirring and the reaction solution was then boiled.Subsequently, the reaction solution was cooled in ice water to prepare asolution of colloidal gold with an average particle diameter of 30 nm.The solution of colloidal gold having an average particle diameter of 30nm was adjusted to 1 OD/mL with a 20 mmol/L Tris buffer solution (pH7.5).

(ii) Preparation of Anti-D-Dimer Antibody Conjugate

To 20 mL of the 1 OD/mL colloidal gold solution (pH 7.5), 1 mL of theanti-D-Dimer monoclonal antibody (Clone #672102) diluted to 60 μL/mL bya 10 mmol/L Tris buffer solution (pH 7.5) was added and stirred at roomtemperature for 10 minutes. To the mixture solution of the colloidalgold and the antibody, 1 mL of the blocking agent N102 (manufactured byNOF Corporation) was added and stirred at room temperature for 5minutes. Subsequently, 2 mL of a 20 mmol/L Tris buffer solutioncontaining 5% (w/v) Blocking Peptide Fragment (manufactured by TOYOBO)was added, further stirred for 5 minutes, and then centrifuged at 10degrees Cat 11900×g for 45 minutes.

After supernatant was removed, 800 μL of a conjugate-diluting solutionwas added to the acquired sediment and suspended to acquire theanti-D-Dimer antibody conjugate.

2) Production of Conjugate Pad

The anti-D-Dimer monoclonal antibody-sensitized conjugate prepared in 1)was mixed with purified water or the following buffer solutions each ofthe water and buffer solutions containing 1.5% Blocking Peptide Fragmentand 2.4% lactose solution to produce a conjugate solution of 22 OD/mL,and a glass fiber pad having a certain volume (Pall Corporation, No.8964) was impregnated with 1.2 volumes of the conjugate solutionrelative to the volume of the pad. The pad was heated and dried at 70degrees C. for 45 minutes in a dry oven to acquire a conjugate pad. Ifan additive such as a sensitizer is added as needed, a necessary amountmay be added to the conjugate solution before performing the sameoperation.

3) Production of Anti-D-Dimer Antibody-Immobilized Membrane

The anti-D-Dimer monoclonal antibody (Clone #DD3B6, manufactured byAmerican Diagnostica) was prepared at 1.5 mg/mL in a 20 mmol/L HEPESbuffer solution (pH 8.0) containing 2.5% sucrose, and a fibrindegradation product was diluted and adjusted to 2.5 mg/mL with a 10mmol/L Tris buffer solution (pH 7.5) containing 2.5% sucrose for thepurpose of coloring of the control line. The anti-D-Dimer monoclonalantibody was applied onto a nitrocellulose membrane (Millipore, HF180,260 mm×25 mm) at a position inside one end of a short side and thefibrin degradation product was applied at an interval of about 5 mm byusing an immunochromatography dispenser “XYZ3050” (BIODOT) set to 0.8μL/cm in a line shape. The membrane was dried at 40 degrees C. for 45minutes in a dry oven to acquire an antibody-immobilized membrane. Thefibrin degradation product is prepared from stabilized fibrin byhydrolysis with plasmin. Color develops when the conjugate sensitizedwith the anti-D-Dimer monoclonal antibody binds to the fibrindegradation product, exhibiting the control line for securing theperformance of an immunochromatographic reagent.

4) Production of Sample Pad

The sample pad was produced by the same operation as Example 1.

5) Production of Immunochromatographic Test Strip

The test strip was produced by the same operation as Example 1. It isnoted that (c) and (d) were replaced with the anti-D-Dimer antibody andthe fibrin degradation product, respectively.

2. Test Results

Study on Type of Buffer Solution Applied to Sample Pad

Each immunochromatographic test strip using a sample pad and a conjugatepad impregnated with each of the nine buffer solutions or purified waterwas used for measuring the level of aggregation of the conjugate aftercontacting with polybrene and spreading over a predetermined time. Aplasma sample collected and centrifuged in an EDTA-2Na blood-collectingtube (Terumo Corporation) supplemented with the fibrin degradationproduct was used as a sample instead of whole blood so as to moreclearly reveal the colloidal gold conjugate-aggregation effect ofpolybrene. To the sample pad window of the test device, 120 μL of thesample was added, and an overlapping portion between the bloodcell-separation film (e) and the antibody-immobilized membrane (b) ofthe test device was uncovered 10 minutes later to measure the reflectedlight intensity by using the immunochromatography reader ICA-1000(Hamamatsu Photonics). The criterion for judgment is the same as Example1.

TABLE 1 Example 1 Example 2 Buffer Aggregation Aggregation solution pHsensitivity Judgment sensitivity Judgment Bis-Tris 5.5 0.0 − 8.3 ± (pKa:6.46) 6.0 0.0 − 5.2 ± 6.5 0.0 − 169.6 ++ 7.0 58.3 + 295.1 ++ MES 5.5 0.0− 5.4 ± (pKa: 6.15) 7.0 4.0 − 61.2 + Citric acid 4.0 0.0 − 0.0 − (pKa:6.4) 5.5 2.2 − 1.5 − (pKa: 4.76) 6.5 0.0 − 4.7 − (pKa: 2.12) Purifiedwater − 38.2 + 48.8 +

When Bis-Tris or MES was used, the effect was confirmed at pH lower thanpKa in the studied pH range. For citric acid, the effect was confirmedin the entire studied pH range.

This is common to Examples 1 and 2 and it is confirmed that even whendifferent materials are used, the effect can be exerted on the samecondition.

INDUSTRIAL APPLICABILITY

According to the present invention, in an immunochromatography methodusing polybrene as a blood-agglutinating agent and a colloidal goldconjugate as a detection reagent, red blood cells can be agglutinatedand then separated and removed from a sample without aggregation ofcolloidal gold by using a certain buffer solution and, therefore, anobject in a sample can accurately be detected and measured. Sinceaddition of a polyanion as a neutralizer is not an essential condition,a reagent configuration can be made extremely simple and, therefore, thepresent invention advantageously leads to reduction of factors causingunpredictable effect to the immune reaction and to the spread of theobject on the strip.

REFERENCE SIGNS LIST

(a) plastic adhesive sheet

(b) antibody-immobilized membrane

(c) anti-H-FABP antibody

(d) anti-KLH antibody

(e) blood cell-separation film (3rd pad)

(f) conjugate pad

(g) sample pad

(h) absorption pad

1. An immunochromatographic detection method, comprising the steps of(A) supplying a sample to a sample-supply portion of a test strip, saidtest strip comprising a membrane, said membrane comprising a porousbody, which comprises a sample-supply portion and a spreading portion,wherein the sample-supply portion is upstream from the spreadingportion, and wherein a conjugate comprising a colloidal gold sensitizedwith an anti-analyte antibody is retained in a dissoluble manner in aconjugate-retaining part of the spreading portion, and an antibody isimmobilized in a detecting part of the spreading portion, which isdownstream from the conjugate-retaining part; (B) agglutinating redblood cells in the sample through contact between hexadimethrine bromideand the sample in the sample-supply portion or in the upstream of thesample-supply portion; (C) separating and removing aggregates acquiredat step (B) from the sample; (D) bringing a sample component acquired atstep (C) after separation and removal of the aggregates into contactwith the colloidal gold-containing conjugate, the step being performedin the presence of a buffer solution component; and (E) detecting acomplex of the analyte in the sample component and the conjugateacquired at the step (D) in the detecting part, and wherein the buffersolution component is selected from the group consisting of Bis-Tris,MES, and citric acid.
 2. The detection method of claim 1, wherein thesample-supply portion comprises hexadimethrine bromide.
 3. The detectionmethod of claim 1, wherein the sample-supply portion contains the buffersolution component for suppressing aggregation of the conjugate due tohexadimethrine bromide.
 4. The detection method of claim 2, wherein thesample-supply portion contains the buffer solution component forsuppressing aggregation of the conjugate due to hexadimethrine bromide.5. The detection method of claim 1, wherein the test strip does notcomprise a polyanion as a neutralizer for neutralizing a cation ofhexadimethrine bromide acting as the blood-agglutinating agent.
 6. Thedetection method of claim 1, wherein the buffer solution component isBis-Tris.
 7. The detection method of claim 1, wherein the buffersolution component is MES.
 8. The detection method of claim 1, whereinthe buffer solution component is citric acid.
 9. The detection method ofclaim 1, wherein the sample-supply portion comprises glass fiber.